Synthesis, Characterization and Antibacterial Activity of some Novel Thiosemicarbazides, 1,2,4-Triazol-3-thiols and their S-substituted Derivatives.

The thiosemicarbazides 3a-c were appeared by reaction of the corresponding substituted hydrazides 1a-c with allylisothiocyanate 2. Synthesis of some novel 1,2,4-triazole-thiols 4a-c bearing a pyridyl unit using 1-(x-picolinoyl)-4-allyl-thiosemicarbazides (x = 2,3,4) in an alkaline solution, is reported. Also, the S-alkylation of triazole derivatives 5-7a-c is described. The structure of the synthesized compounds resulted from the IR, 1H and -13C NMR spectroscopy and elemental analysis data. The antibacterial studies to all of the synthesized compounds against B. cereus, E. coli, P. aeroginosa, S. aureus and E. faecalis as MIC values are reported. Some of these compounds such as 7a, 4a and 3a exhibited a good to significant antibacterial activity.


Introduction
It is well known that thiosemicarbazide derivatives exhibit interesting biological properties such as antitubercular (1), antiviral (2), antimalarial and antibacterial activity (3). These compounds are not only used as a segment in biologically and chemically buildings (4-6), but also as a versatile intermediate for the synthesis of importance heterocycles such as triazoles, thiadiazoles, oxadiazoles and thiazolidinones (7-9).
In view of these reports and as part of ongoing studies on the synthesis and biological consideration of heterocycles (22,23,25,26), we wish to describe the synthesis of new series of 5-membered heterocyclic titled compounds, bearing the 5-isomeric pyridyl, 4-N-allyl and their antibacterial activities. 256,128,64,32,16,8 μg/mL were used for all bacteria in per disc and incubated the same conditions.

Experimental
All chemicals used were purchased from Merck or Fluka. Melting points were determined using an electrothermal digital apparatus and are uncorrected. FT-IR spectra were obtained with a SHIMADZU -IR Presting-21 spectrometer using KBr discs. NMR spectra were recorded on a Bruker (400 or 500 MHz) spectrometer. Chemical shifts (ppm) are referenced to tetramethylsilane (TMS) as internal standard. Elemental analyses were performed with as Elemental Analyzer (Elemental, Vario EL III) at Arak University. Reactions were monitored by thin layer chromatography (TLC). The 2-pyridyl hydrazide 1a was prepared with low improvement following the previously reported procedure (27).

Biological screening
The antibacterial activity of synthesized compounds were screened at a concentration of 5 mg/mL against five reference strains of bacteria (Three gram positive as Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, and two gram negative as Escherichia coli ATCC 25922, pseudomonas aeruginosa ATCC 27853). Tested compounds were dissolved in dimethyl sulfoxide (DMSO) for the preparation of stock solution. The solvent control was included, although no antibacterial activity has been noted. All samples were tested in triplicate and the average results were recorded. Microbial susceptibility testing of all compounds was carried out by diffusion agar and minimal inhibitory concentration (MIC) methods according to Clinical and Laboratory Standards Institute (CLSI) guideline (28). The suspension of bacteria was adjusted to 0.5 MacFarland Standard (10 6 c.f.u/mL) and spread over Muller-Hinton Agar. The tested compounds are placed in random well position on the plate, after overnight incubation at 37 °C the zone of inhibition determinate. The antibacterial effects of the compounds that produced ≥ 8mm zone of inhibition were tested quantitatively by Micro broth dilution method for determination of MIC value that were defined as lowest concentration of compound for inhibition growth of tested bacteria. In this method concentration of 512, Hz, H pyr ), 9.13 (s, 1H, H pyr ), 9.93 (br, 1H, NH) ppm; 13 C-NMR (acetone-d 6, 100MHz): δ = 46.4 (C-aliphatic), 115.0, 123.4, 128.2, 134.5, 135.2, 148.8, 152.6 (C-Allyl and Aryl), 165.1 (C=S), 183.7 (C=O) ppm; Anal. Calcd. for C 10 H 12 N 4 OS: C, 50.83; H, 5.12; N, 23.71; S, 13.57; found: C, 50.47; H, 5.14; N, 23.65; S, 13.52%.

General procedure for the synthesis of 1,2,4-triazole-3-thiols 4(a-c)
A solution of thiosemicarbazide, 3(a-c), (5 mmol, 1.180 g) in 2N NaOH (10 mL) was refluxed for 2-3 h. The resulting solution was cooled to room temperature and acidified (pH = 3) with 2N HCl. The precipitate was filtered and washed with water and ethanol. The obtained compound was dried and crystallized from suitable solvent to give compound 4(a-c). 4

Synthesis
The synthesis of the titled compounds is illustrated in Figure 1. The preparation of thiosemicarbazides 3a-c was achieved by reaction of the isomeric pyridine carboxylic acid hydrazides 1a-c with allylisothiocyanate 2. Thiosemicarbazide derivatives 3a-c underwent an intramolecular cyclization under basic conditions to produce 1,2,4-triazoles 4a-c in high yields. The reaction yields for 4c (94%) is higher than other respective isomers, which may attributed to the stereo-electronic effects of the nitrogen atom of the pyridine ring and also due to the more symmetrical structure of these products. The S-alkylated 1,2,4-triazoles, esters 5a-c, acetamides 6a-c and acetic acids 7a-c were also prepared by reaction of 1,2,4-triazole-3-thioles 4a-c and corresponding reagents, as previously reported (6,21).
The IR spectra of thiosemicarbazides 3a-c showed characteristic absorption bonds at   C NMR spectra of 4a-c, the appearance of signals at the region 168.5 ppm attributed to the carbon resonance of the C=N or C=S group in triazole rings which is in support of the expected structures.
In the 1 H NMR spectra of 5-7a-c, the absence of the -SH resonance and the appearance of a singlet in the aliphatic region, related to the resonance of the -SCH 2 -group, supports the formation of the alkylated products.  (1) (3) in exhibited region of the spectrums. However, the acidic proton (COOH) of compounds 7a-c was not observed in the spectrum, probably due to the effect of exchange of this acidic proton with deuterium of small amounts of D 2 O, which is present in DMSO-d 6 , as a solvent (29).

Antibacterial activities
Applying the agar plate diffusion technique (30), all of newly synthesized compounds were screened in-vitro for antimicrobial activities against five pathogenic bacteria. The results of the bioassay are given in Table 1. A cursory view of the data indicates that some of the compounds 4,6,7a and specially 3a exhibit a moderate to good activity against four bacteria with gram positive (Bacillus cereus, Staphylococcus aureus) and negative (Escherichia coli, pseudomonas aeruginosa) strain. It is considerable that thiosemicarbazide 3a was found to be more active against three microorganisms than Gentamicin, which is a known antimicrobial drug. Therefore, this compound can have the potential to be good antibacterial candidate that the research is ongoing in this regard. Although compounds 7a and 7c has shown the good activity against E. faecalis, the other compounds do not show any antibacterial activity.
In addition, investigation of the minimum inhibitory concentration (MIC) values of the potent derivatives against five microorganisms was performed and the results are presented in Table 2. Just as was predicated, compound 3a indicates the highest bactericidal activity (16-64 µg/mL) against all organism tests (except for E. faecalis).